Breuer Cort B, Xiong Zhewen, Wang Alice, Rodriguez Grayson E, Abhiraman Gita C, Garcia K Christopher, Reticker-Flynn Nathan E
Immunology Program, Stanford University, Stanford, CA, USA.
Department of Otolaryngology - Head & Neck Surgery, Stanford University, Stanford, CA, USA.
Nat Protoc. 2025 Aug 13. doi: 10.1038/s41596-025-01200-5.
Lymph node (LN) metastasis is a conserved feature across most solid organ malignancies and portends worse prognoses. Functionally, LN metastases induce systemic tumor-specific immune tolerance and may serve as a reservoir for distant metastases. Nonetheless, there are relatively few preclinical models for interrogating the biology of LN metastasis and its systemic effects at various stages of metastatic progression. We describe a method for modeling LN metastasis of melanoma tumors in mice that enables assessment of tumor and immune cell phenotypes and the functional roles of nodal involvement on distant metastasis. Our model comprises a family of transplantable syngeneic melanoma tumor cell lines evolved to exhibit enhanced LN metastatic potential, which can be used to probe cancer-immune interactions and test new therapeutics. We present both (i) a spontaneous LN metastasis model involving primary tumor implantation and assessment of LN colonization 21-28 d later and (ii) an experimental metastasis model involving implantation of primary tumors followed by direct intra-LN injections of tumor cells. Both models can be extended to assess the impact of LN metastasis on the development of distant metastases through asynchronous intravenous injections of tumors. Finally, we discuss experimental design considerations including when to use spontaneous or experimental models and troubleshooting consistent LN metastasis, making this model accessible for researchers with basic mouse survival-surgery skills. We highlight how LN metastasis models can be used to profile metastatic immune reprogramming, measure the impact of nodal metastases on distant metastases and assess novel anti-metastatic therapeutics.
淋巴结（LN）转移是大多数实体器官恶性肿瘤的一个共同特征，预示着更差的预后。在功能上，LN转移会诱导全身性肿瘤特异性免疫耐受，并可能成为远处转移的一个病灶。尽管如此，用于探究LN转移生物学及其在转移进展各个阶段的全身效应的临床前模型相对较少。我们描述了一种在小鼠中模拟黑色素瘤肿瘤LN转移的方法，该方法能够评估肿瘤和免疫细胞表型以及淋巴结受累对远处转移的功能作用。我们的模型包括一系列可移植的同基因黑色素瘤肿瘤细胞系，这些细胞系经过进化以表现出增强的LN转移潜能，可用于探究癌症与免疫的相互作用并测试新的治疗方法。我们展示了两种模型：（i）一种自发LN转移模型，涉及原发性肿瘤植入并在21 - 28天后评估LN定植情况；（ii）一种实验性转移模型，涉及原发性肿瘤植入，随后直接向LN内注射肿瘤细胞。这两种模型都可以扩展，通过异步静脉注射肿瘤来评估LN转移对远处转移发展的影响。最后，我们讨论了实验设计方面的考虑因素，包括何时使用自发或实验模型以及解决LN转移一致性的问题，使具备基本小鼠生存手术技能的研究人员能够使用该模型。我们强调了LN转移模型如何可用于分析转移性免疫重编程、测量淋巴结转移对远处转移的影响以及评估新型抗转移治疗方法。
